Hair follicle stem cell-specific PPARgamma deletion causes scarring alopecia

Abstract

Primary cicatricial or scarring alopecias (CA) are a group of inflammatory hair disorders of unknown pathogenesis characterized by the permanent destruction of the hair follicle. The current treatment options are ineffective in controlling disease progression largely because the molecular basis for CA is not understood. Microarray analysis of the lymphocytic CA, Lichen planopilaris (LPP), compared to normal scalp biopsies identified decreased expression of genes required for lipid metabolism and peroxisome biogenesis. Immunohistochemical analysis showed progressive loss of peroxisomes, proinflammatory lipid accumulation, and infiltration of inflammatory cells followed by destruction of the pilosebaceous unit. The expression of peroxisome proliferator-activated receptor (PPAR) gamma, a transcription factor that regulates these processes, is significantly decreased in LPP. Specific agonists of PPARgamma are effective in inducing peroxisomal and lipid metabolic gene expression in human keratinocytes. Finally, targeted deletion of PPARgamma in follicular stem cells in mice causes a skin and hair phenotype that emulates scarring alopecia. These studies suggest that PPARgamma is crucial for healthy pilosebaceous units and it is the loss of this function that triggers the pathogenesis of LPP. We propose that PPARgamma-targeted therapy may represent a new strategy in the treatment of these disorders.

Figure 1. Histology of normal and LPP scalp tissue

(a) Structure of the human hair follicle and the sebaceous glands (the pilosebaceous unit). The hair follicle stem cells are located in the hair follicle bulge region (b) between the arrector pili muscle (APM) and the sebaceous gland (SG). Other parts of the hair follicle depicted are the outer root sheath (ORS), inner root sheath (IRS), bulb (Bb), dermal papilla (DP), and matrix (M). (b) In scarring alopecias, inflammation occurs in the permanent portion of the hair follicle (infundibular region; striped area). Hematoxylin and eosin (H&E) staining of scalp biopsy sections (region of sebaceous glands) of normal at (c; × 10) and (d) Well-formed pilosebaceous units (× 20). LPP tissue sections (infundibular region) at (e; × 10) and (f; × 20) have very few hair follicles, atrophied sebaceous glands, and dense lymphocytic infiltrate around many hair follicles. Scale bar = 25 µm.

Figure 2. Peroxisome deficiency in LPP

Staining of peroxisomes with the anti-PMP-70 primary antibody for the peroxisomal membrane protein and Alexa 488-labeled secondary antibodies. In normal scalp tissue (normal), a characteristic “punctate” pattern of peroxisome staining is seen specifically in the sebaceous glands (SG) and in the ORS and IRS surrounding the hair shaft (HS). In unaffected tissue (uninvolved), peroxisome staining is lost in the ORS and IRS cells surrounding the hair shaft but not in sebaceous glands. Double staining of the unaffected tissue sections for peroxisomes and nuclei with (DAPI) shows that the ORS and IRS cells around the hair shaft are intact but the cells have lost peroxisomes (uninvolved, lower panel). Affected LPP tissue (LPP) shows a complete lack of staining for peroxisomes in the sebaceous glands and in the ORS and IRS cells surrounding the hair shaft. In LPP tissue, peroxisome staining is not seen in both the sebaceous gland and the hair follicle. Double staining the tissue for peroxisomes and nuclei reveals the presence of an intact sebaceous gland and hair follicle in LPP tissue (right, lower panel), suggesting the specific loss of peroxisomes. Scale bar = 25 µm.

Figure 3. Surface plot analysis of peroxisomes in normal scalp tissue sections and in LPP

High-magnification (× 40) confocal microscopy of (a) normal scalp sections, stained for peroxisomes with the anti-PMP-70 primary antibody, yielded a punctate pattern indicating the presence of peroxisomal membranes and intact peroxisomes. In LPP (b), no PMP-70-positive particles were visible indicating the complete absence of intact peroxisomes (c). Normal scalp section minus primary antibody control showed an absence of peroxisomal staining. Three-dimensional graphs (d–f) of the intensities of pixels in the peroxisomal images were determined by surface plot analysis with the NIH software Image J. The intensity of peroxisomal staining within a defined region (white rectangular areas highlighted in a–c) is interpreted as height for the plot and gives a comparative semiquantitative measure of the number of peroxisomes within the region in (d) normal, (e) LPP tissue and (f) normal minus primary antibody control. Scale bar = 25 µm.

Figure 4. Altered PPARγ gene expression in LPP and its effect on PEX and COX2 genes

(a) PPAR gene expression in normal and LPP tissue. Total RNA was isolated from normal control (N = 10), unaffected LPP (N = 10), and affected LPP scalp tissue (N = 10) and PPARγ, PPARα, and PPARδ gene expression was measured by real-time PCR. PPARγ expression was significantly decreased in unaffected and affected LPP tissue. However, the expression of PPARα and PPARδ remained unchanged in LPP compared to control samples. (b) PPARγ modulation affects PEX16 gene expression in vitro. HaCaT keratinocytes were treated with vehicle alone (0.1% DMSO) or with specific agonists (1 and 5 µm in 0.1% DMSO) of PPARγ-Ciglitazone (Cig), Rosiglitazone (Rosi), Pioglitazone (Pio), and Troglitazone (Tro), PPARα-WY-14363 and PPARδ–GW50516. After 48 hours, PEX16 gene expression was measured by real-time PCR. PPARγ agonists significantly induced the expression of PEX16 gene; however, PPARγ and PPARδ agonists had minimal effect. *represents P value ≤0.005. (c) PPARγ modulation affects PEX3 gene expression in outer root sheath (ORS) keratinocytes in vitro. In ORS cells, PPARγ agonist (Pioglitazone) significantly induced PEX3 gene expression compared to vehicle alone (0.1% DMSO). In contrast, PPARα–(WY-14363) and PPARδ (GW50516) agonists had minimal effect, suggesting that PPARγ regulates peroxisome biogenesis in the pilosebaceous units. (d) PPARγ agonist (Pio) significantly inhibited COX2 gene expression in ORS whereas PPARα–(WY-14363) and PPARδ (GW50516) agonists had minimal effect, suggesting that PPARγ negatively regulates COX2 expression.

Figure 5. Targeted disruption of PPARγ in stem cells of the bulge resulted in scarring alopecia

(a) and (b) PPARγ^fl/fl/Cre mice (females, 3 months) with PPARγ^fl/fl littermate (male). Hair loss occurs in a random patchy and progressive manner. The mice display severe scratching behavior and are smaller than their normal littermates. (c) A close-up of the PPARγ^fl/fl/Cre mice shows a region with absence of follicular markings (indicated by arrow) and erythema suggesting the presence of inflammation. The PPARγ^fl/fl littermates have normal skin and hair phenotype. Histology of skin of PPARγ^fl/fl and PPARγ^fl/fl/Cre mice by Hematoxylin and eosin (H&E) staining of (d) PPARγ^fl/fl mice shows normal hair follicles and sebaceous glands. (e) H&E staining of a PPARγ^fl/fl/Cre mouse shows hyperkeratosis and follicular plugging (indicated by arrow). The sebaceous glands appeared normal in the early stage disease (2–3 months). Scale bar = 50 µm.

Figure 6. PPARγ^fl/fl/Cre mice show histopathological features of scarring alopecia

H&E staining of the skin of PPARy^fl/fl/Cre mice showed (a) dystrophic hair follicles, (b) follicular plugging, (c) dystrophic sebaceous glands, Scale bar = 20 µm, (d) interstitial, and (e) perifollicular inflammation. Scale bar = 50 µm.

Figure 7. PPARγ^fl/fl/Cre mouse skin shows scarring

H&E staining showed (a) normal hair follicles in wild-type mouse skin (b) Dystrophic hair follicle with sebaceous gland atrophy in the skin of PPARγ^fl/fl/Cre mice; (c–e) follicular scarring, in which fibrous connective tissue strands run perpendicular to the epidermis from remnants of dystrophic hair follicles in PPARγ^fl/fl/Cre mouse >4 months of age (H&E; × 40). Scale bar = 50 µm.

Figure 8. Proposed model for the pathogenesis of primary cicatricial alopecia

In normal pilosebaceous units, PPARγ binds to PPAR response elements (PPREs) on target genes and maintains lipid homeostasis by regulating peroxisome biogenesis and lipid metabolism. PPARγ also modulates the inflammatory response by regulating the expression of cytokine genes. In primary CA, PPARγ deficiency (either because of environmental or genetic factors) causes loss of peroxisome biogenesis, deregulates lipid metabolism, and produces proinflammatory lipids that trigger an inflammatory response that in turn causes tissue damage and permanent hair loss in CA.